【作者】 欧阳志荃；

【导师】 胡志红； 孙修炼；

【作者基本信息】 中国科学院研究生院(武汉病毒研究所) ， 生物化学与分子生物学， 2003， 硕士

【摘要】 棉铃虫单核衣壳核多角体病毒（Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus，HaSNPV）能专一性地感染棉铃虫，作为生物杀虫剂已得到广泛应用，本论文用免疫组织化学的方法研究HaSNPV感染棉铃虫幼虫的组织病理变化，深入了解 HaSNPV的致病机理及其与宿主的相互关系，同时对HaSNPV的组织蛋白酶基因开展功能研究，论文包括三章。第一章首先对杆状病毒的研究状况作了综述报道，主要包括杆状病毒的分类研究、形态结构、细胞病理、组织病理、与侵染宿主相关基因以及对宿主的系统感染过程等。另一部分介绍了本论文研究的背景,包括HaSNPV的研究历史，当前的分子生物学研究进展，HaSNPV的病理研究以及本论文的研究目的和意义。第二章报道了HaSNPV感染棉铃虫幼虫的病理时相及HaSNPV多角体蛋白在幼虫组织中表达的免疫组化研究。以5×103 PFU的HaSNPV出芽病毒粒子（BV）注射4龄初的棉铃虫幼虫，免疫组化结果表明，感染后24小时，未能检测到多角体蛋白在幼虫组织中的表达；感染后48小时，多角体蛋白可在部分脂肪体、气管组织、血细胞和真皮组织的细胞核中表达；72小时后，被感染的脂肪体、气管组织、和真皮组织的细胞数比48小时多； 96小时后，多角体蛋白可以在脂肪体、气管、真皮组织中大量表达，48小时到96小时间，被感染的血细胞数目基本不变，中肠组织和肌肉都未检测到多角体蛋白的表达；幼虫死亡后，可在中肠上皮细胞的基底膜间隙中检测到多角体蛋白的表达，肌肉组织中未见表达信号，其它组织全部被感染并且组织结构被破坏。H.E染色的结果与免疫组化的结果基本相符。第三章对HaSNPV的组织蛋白酶基因（v-cath）进行了初步研究。棉铃虫核多角体病毒的组织蛋白酶基因共1，098个碱基，预测蛋白质的大小为42 kDa。RT-PCR结果显示，v-cath 的转录开始于感染后12小时，一直延续到96小时以后。3’RACE表明v-cath的转录本在翻译终止密码子TAA下游23 nt处加有PolyA尾。我们对v-cath 进行截短型原核表达，获得表达产物并制备了相应的多克隆抗体，并检测了抗体的特异性。同时对<WP=5>缺失v-cath的病毒进行组织病理研究，结果显示缺失组织蛋白酶基因的病毒感染的幼虫组织明显保持比较完整和致密，且死后虫体不液化。本实验结果证明v-cath是与组织液化相关的基因。更多还原

【Abstract】 Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV) can specifically infect the Helicoverpa armigera larvae and now it has been widely used as a biopesticide in China. The pathogenesis process of Helicoverpa armigera larvae infected with HaSNPV was studied using H.E staining and immunohistochemistry in order to understand the mechanism of the infection and the interaction between the virus and its host. We also studied the cathepsin gene of the HaSNPV. The thesis includes three chapters. Chapter one is a general introduction of the research on baculovirus, it includes the overview of baculovirus, taxonomy, structure, cellular pathology, tissue pathology of the baculovirus, the genes related to the infection and pathology, and the system infection process. The background of the research and the aim of this thesis are also included. Chapter two reported the pathogenesis process of Helicoverpa armigera larvae infected with HaSNPVusing H.E staining and immunohistochemistry with antibody against polyhedrin. The fourth instars Helicoverpa armigera larvae were injected with 5×103 PFU HaSNPV per larvae. The immunohistochemistry results indicated the polyhedrin protein could not be detected at 24 h.p.i. . At 48 h.p.i., it was found partially expressed in the tissues of fat body, trachea epidermis and epidermis. From 72 to 96 h.p.i., the polyhedrin was found heavily expressed in fat body, trachea epidermis and epidermis. No polyhedrin was found expressed in midgut untill larvae dead and the muscule was not infected at all the time points.V-cathepsin gene(v-cath) is related to the liquefaction of the host in other baculovirus and in chapter three we characterized the cathepsin gene of HaSNPV. Its ORF is 1089 bp, encode a protein about 42kDa. RT-PCR showed that the transcription of the v-cath begins at 12 h.p.i. and sustained to 96 h.p.i., a poly(A) tail was found at 23nt downstream of the stop codon TAA by 3’RACE. To detect the translation product, we expressed truncated C terminus of V-cath in E. coli and generated specific antibody. The his-pathology of the recombinant virus with the v-cath gene deletion was presented and the result showed that v-cath of HaSNPV is related to host liquation.更多还原